DESCRIPTION (from the application): This laboratory has been a major contributor to our understanding of the mechanism of rRNA transcription initiation and regulation. However, the experimental system we have studied does not exhibit some characteristics of higher eukaryotic rRNA transcription, in particular, it does not require an upstream activator or upstream promoter elements. Because S. cerevisiae does not require these, and further has very facile and powerful genetics, a biochemical and genetic study of yeast is proposed. Over the past decade, the genetics of rRNA transcription has been effectively exploited in yeast, with isolation of genes defining three transcription factors, UAF, CF, and Rrn3p, which are needed in addition to polymerase I and TATA-binding factor for activated transcription. The biochemistry of the system has lagged significantly behind the genetics, leaving a number of mechanistic questions unanswered. These will be investigated. The structure of the committed and preinitiation complexes will be revealed by a battery of approaches, including five footprinting methods and site-specific DNA-protein photo-cross-linking, drug inhibition studies, DNA topological analyses and genetic screens for TBP mutants. These studies will determine the architecture of the preinitiation complexes, determine the groove of the DNA primarily contacted by proteins, and the path of the DNA in these complexes. Yeast two-hybrid analyses will reveal the protein-protein interactions in the complexes. The mechanistic role of TBP in activated transcription will be elucidated, with emphasis on its mechanism of interaction with DNA, and resulting DNA topology changes. In addition, the functional mechanism of several of these factors will be investigated. In particular, the mechanism of the transcription factor implicated in the regulation of rRNA transcription, Rrn3p, will be determined; assays to determine if it mediates polymerase recruitment, DNA melting or promoter clearance will be used. The RNA polymerase I and the transcription factor subunits that interact with each other to mediate pol I recruitment will be determined both biochemically and genetically. The potential role of polymerase subunit or Rrn3p modification in regulation will be investigated. These studies will yield the most complete description of the biochemistry and genetics of any eukaryotic rRNA transcription system.